Nanopore sequencing indicates that tandem amplification of chromosome 20q11.21 in human pluripotent stem cells is driven by break induced replication

Copy number variants (CNVs) are genomic rearrangements implicated in numerous congenital and acquired diseases, including cancer. The appearance of culture-acquired CNVs in human pluripotent stem cells (PSC) has prompted concerns for their use in regenerative medicine applications. A particular problem in PSC is the frequent occurrence of CNVs in the q11.21 region of chromosome 20. However, the exact mechanism of origin of this amplicon remains elusive due to the difficulty in delineating its sequence and breakpoints. Here, we have addressed this problem using long-read Nanopore sequencing of two examples of this CNV, present as a duplication and as a triplication. In both cases, the CNVs were arranged in a head-to-tail orientation, with microhomology sequences flanking or overlapping the proximal and distal breakpoints. These breakpoint signatures point to a mechanism of microhomology-mediated break-induced replication in CNV formation, with surrounding Alu sequences likely contributing to the instability of this genomic region.